Although great progress has been made in the study of metabolism of vitamin D3, studies on the metabolism of vitamin D2 have been severely limited by a lack of 25-hydroxyvitamin D2 and 1,25-dihydroxyvitamin D2. Methods will be developed for the chemical synthesis of 25-hydroxyvitamin D2 and 1,25-dihydroxyvitamin D2 to provide adequate quantities for biological testing and as reference standards for the assay of these two important metabolites in blood and tissues of experimental animals or humans. The development of these syntheses will also make available tritium-labeled 25-hydroxyvitamin D2 and tritium-labeled 1,25-dihydroxyvitamin D2 of high specific activity suitable for studies of metabolism and for use in the assay of these materials in biological fluids. Finally, the proposed synthetic work will yield intermediates from which a series of 24(R) or (S)-alkyl vitamin D2 analogs can be prepared in a very simple manner. All synthetic compounds will be extensively tested for biological activity in rats and chicks. In rats the ability of these compounds to mineralize bone, stimulate intestinal calcium transport, and mobilize calcium from bone will be examined. Similar parameters will be measured in the chicken. Completion of this work would provide much needed standards for analysis of the biologically important vitamin D2 metabolties and would make available essential material for many fruitful investigations in the vitamin D field, especially those focussing on the D2-series.